SUMMARY for TR&D 2 TR&D 2 of the BTRC will develop novel synthetic, non-metallic reporter genes that can be detected with chemical exchange saturation transfer (CEST) MRI for precise visualization and pinpointing of biological processes in living organisms. Using a lysine-rich protein (LRP) as such a prototype gene, we have previously demonstrated that 1) We can image rapidly dividing tumor cells without the limitation of a label dilution effect that currently exists with conventional MR contrast agents; 2) We can image promoter-driven specific gene expression; and 3) we can image oncolytic virotherapy. In this TR&D, we aim to dramatically improve the CEST contrast and biocompatibility of LRP for further dissemination to the scientific community, and to create a pathway towards eventual clinical translation. Using advanced, rational design-based molecular-genetic engineering approaches, we will first develop a so-called ?enhanced? LRP, or eLRP (Aim 1a). Enhancement is defined by transcription and translation efficiency, protein refolding, optimal proton exchange rate, and strongest CEST contrast. For the latter, a custom-designed high-throughput screening methodology will be used to determine optimal peptide sequence configurations. Next, we will ?humanize? eLRP to create heLRP, using an array of immunological assays (Aim 1b). We will use established algorithms to identify epitopes that induce a T cell and/or humoral response in reverse. Re-engineered LRPs will undergo reiterated screening processes until all immunogenicity has been eliminated without compromising CEST contrast. Alternatively, we will use human protamine-1 (hPRM1) as a starting template to create chimeric LRP/hPRM1 constructs through DNA shuffling. The absence of serum polyclonal antibodies from heLRP-immunized rabbits will be used as a final key criteria for TR&D 2 dissemination. During this immunological screening process, we will simultaneously identify the counterpart of heLRP, i.e., an ?immunogenic? LRP or iLRP. Following in vivo transfection, this iLRP will be used as a new theranostic vector to simultaneously induce an anti-tumor immune response and visualize subsequent tumor cell regression (Aim 2). Finally, we aim to demonstrate how eLRP can be used to provide a unique dynamic insight into biological processes and as defined by cell-cell interactions. We have chosen dendritic cell (DC) immunotherapy as an example. Following a validation study to confirm that constitutively expressed eLRP DCs can be detected in vivo when migrating to lymph nodes following vaccination (Aim 3a), we will investigate when and where DC activation occurs upon presenting antigen to CD4+ cells. We aim to accomplish this using IL-12 promoter-driven specific expression (Aim 3b). Concurrently, we will assess the time course and biodistribution of activated, Ova-specific CD4+ transgenic cells using BLI in an Ova-expressing melanoma mouse model. Our LRP reporters will have many applications in the study of basic cell biology and cell malfunctioning in a wide variety of disease models, as they can be designed de novo and in silico, and hence have unlimited potential for manipulation and fine-tuning as needed for the precise visualization of the biological process in question.